T.R. Arun

New closed tube loop mediated isothermal amplification assay for prevention of product cross-contamination

Graphical abstract

Loop-mediated isothermal amplification (LAMP) test for specific and rapid detection of Brucella abortus in cattle

Background: Brucella abortus, the major causative agent of abortion in cattle and a zoonotic pathogen, needs to be diagnosed at an early stage. Loop-mediated isothermal amplification (LAMP) test is easy to perform and also promising to be adapted at field level. Objective: To develop a LAMP assay for specific and rapid detection of B. abortus from clinical samples of cattle. Methods: LAMP primers were designed targeting BruAb2_0168 region using specific software tool and LAMP was optimized. The developed LAMP was tested for its specificity with 3 Brucella spp. and 11 other non-Brucella spp. Sensitivity of the developed LAMP was also carried out with known quantity of DNA. Cattle whole blood samples and aborted fetal stomach contents were collected and used for testing with developed LAMP assay and results were compared with polymerase chain reaction (PCR). Results: The developed LAMP assay works at 61 °C for 60 min and the detection limit was observed to be 100-fold more than the conventional PCR that is commonly used for diagnosis of B. abortus. Clinical sensitivity and specificity of the developed LAMP assay was 100% when compared with Rose Bengal plate test and standard tube agglutination test. SYB® green dye I was used to visualize the result with naked eye. Conclusion: The novelty of the developed LAMP assay for specifically detecting B. abortus infection in cattle along with its inherent rapidness and high sensitivity can be employed for detecting this economically important pathogen of cattle at field level as well be exploited for screening of human infections.

In Silico Identification of Diagnostic Candidates from Predicted Lipoproteome of Mycoplasma mycoides subsp. capri

| Mycoplasma mycoides subspecies capri (Mmc), the causative agent of caprine pleuro pneumonia/contagious agalactia is a major pathogen affecting goats worldwide. Development of specific immunodiagnostic assays for Mmc is often hampered by interspecies cross-reactivity with other caprine mycoplasmas and the intra species antigenic variability. The study presents a comparative and subtractive proteomic analysis to identify specific, conserved and immunogenic lipoproteins from Mmc proteome. Analysis of 896 proteins of Mmc strain 95010 predicted 72 lipoproteins by lipoP1.0 server. BLAST analysis revealed 17 putative non cross-reactive lipoproteins out of which seven were found to be conserved within the species. Further computational workflow employing ExPASy protein analysis tools and B cell epitope prediction softwares identified five lipoproteins suitable for development of immunodiagnostics.